Agents and methods for treating and preventing seborrheic keratosis

ABSTRACT

Provided herein are methods and assays for isolating and culturing seborrheic keratosis cells ex vivo. Also provided herein are screening assays using cultured seborrheic keratosis cells and methods for treating seborrheic keratosis in a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/395,737, filed on Oct. 20, 2014, which is a 35 U.S.C. 371 NationalStage Application of International Application No. PCT/US2013/038358filed on Apr. 26, 2013, which designates the United States, and whichclaims benefit under 35 U.S.C. §119(e) of the U.S. provisionalapplications No. 61/638,684 filed on Apr. 26, 2012, the contents of eachof which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Human skin is continuously exposed to an onslaught of environmentalstress, the most significant of which is ultraviolet (UV) light¹⁻³.Chronic exposure to UV radiation leads to oxidative overload andirreparable DNA damage of the cell, which results in altered metabolismand multiple genomic aberrations in epidermal cells. The biologicalconsequences of these processes are accelerated aging and benign, aswell as malignant tumor formation⁴. While it is widely accepted thatmalignant transformation is the result of accumulating genomicalterations in oncogenes and tumor suppressor genes, much less is knownabout the ethology and genetic changes in benign tumors.

In both clinical and experimental situations, the majority of benigntumors fail to progress into malignancy for reasons that are poorlyunderstood. The skin provides an intensely studied model ofself-renewing epithelial tissues, with distinct stem cell populationsgiving rise to tumors with different behavior. Among these, skinsquamous cell carcinomas are among the most frequent human cancers.Besides malignant tumors, keratinocyte subpopulations can give rise tobenign skin tumors like seborrheic keratoses (SKs). These are verycommon lesions that develop with age in the vast majority of the humanpopulation. Common histological features are acanthosis, papillomatosisand hyperkeratosis along with varying degree of pigmentation. SKs haveclinical similarities to the common wart. While human papillomaviruses(HPV) have been implicated in the origin of these lesions, recentanalyses have generally discounted a role for this virus in the majorityof cases. Patients can often have multiple SKs, and individualsdeveloping a great number of these lesions (>50) on a familial basishave been described. In addition, SKs appear to be clonal in origin,indicating that they do not result from a reactive epidermalhyperplasia, but from clonal expansion of somatically mutated cells. Infact, recent work has shown the presence of activating mutations in aspecific transmembrane tyrosine kinase receptor, fibroblast growthfactor receptor-3 (FGFR3) in a large fraction of sporadic SKs. Acausative role of FGFR3 mutations is suggested by the fact thattransgenic mice with keratinocyte-specific expression of an activatedform of the receptor produce skin lesions histologically similar to SKs.

SUMMARY OF THE INVENTION

The methods and assays provided herein are based, in part, on thediscovery of a novel method for isolating and culturing seborrheickeratosis cells ex vivo, a technique that permits study of these cellsin culture. Prior to this discovery, such methods for culturing primaryseborrheic keratosis cells were not available in the art. Also providedherein are screening assays using cultured seborrheic keratosis cellsand methods for treating seborrheic keratosis in a subject.

Provided herein in one aspect is a method for treating a seborrheickeratosis in a subject, the method comprising administering atherapeutically effective amount of a composition that inhibits the Aktsignaling pathway to a subject having a seborrheic keratosis.

In one embodiment of this aspect and all other aspects described herein,the composition is applied topically or administered systemically.

In another embodiment of this aspect and all other aspects describedherein, the method further comprises a step of diagnosing the subjectwith a seborrheic keratosis.

In another embodiment of this aspect and all other aspects describedherein, the therapeutically effective amount of the composition does notsubstantially affect the survival of normal keratinocytes.

In another embodiment of this aspect and all other aspects describedherein, the composition comprises a small molecule, a peptide inhibitor,or an RNAi molecule.

In another embodiment of this aspect and all other aspects describedherein, the composition is an Akt-1 and/or an Akt-2 inhibitor (e.g.,C8).

In another embodiment of this aspect and all other aspects describedherein, the composition further comprises a pharmaceutically acceptablecarrier.

Another aspect provided herein relates to a method for inducingapoptosis in a seborrheic keratosis cell, the method comprisingcontacting a seborrheic keratosis cell with an effective amount of acomposition that inhibits Akt signaling, thereby inducing apoptosis inthe cell.

In one embodiment of this aspect and all other aspects described herein,the effective amount of the composition does not substantially affectthe survival of normal keratinocytes.

In another embodiment of this aspect and all other aspects describedherein, the composition comprises a small molecule, a peptide inhibitor,or an RNAi molecule.

In another embodiment of this aspect and all other aspects describedherein, the composition is an Akt-1 and/or an Akt-2 inhibitor and/or apan-Akt inhibitor.

Another aspect provided herein relates to a method for culturingseborrheic keratosis cells ex vivo, the method comprising: (a)contacting a biological sample comprising seborrheic keratosis cellsobtained from a subject with a solution comprising a dispase enzyme at atemperature and for a time sufficient to initiate dissociation of theseborrheic keratosis cells from the biological sample, and (b) culturingthe dissociated seborrheic keratosis cells.

In one embodiment of this aspect, the method comprises culturingseborrheic keratosis cells ex vivo, the method comprising: (a)contacting a biological sample comprising seborrheic keratosis cellsobtained from a subject with a solution comprising initially a dispaseenzyme and subsequently a trypsin enzyme at a temperature and for a timesufficient to dissociate the seborrheic keratosis cells from thebiological sample, and (b) culturing the dissociated seborrheickeratosis cells.

In one embodiment of this aspect and all other aspects described herein,the temperature is below a standard room temperature of 21° C.

In another embodiment of this aspect and all other aspects describedherein, the time sufficient to initiate digestion of the seborrheickeratosis cells is at least 15 hours. In another embodiment, the timesufficient to initiate digestion of the seborrheic keratosis cells withdispase is at least 15 hours.

In another embodiment of this aspect and all other aspects describedherein, the method further comprises a step of contacting the biologicalsample comprising seborrheic keratosis cells with an additionalprotease.

In another embodiment of this aspect and all other aspects describedherein, the additional protease is Trypsin.

In another embodiment of this aspect and all other aspects describedherein, the method further comprises a step of adding a culture mediumand filtering larger particles from the dissociated cells before theculturing of step (b).

In another embodiment of this aspect and all other aspects describedherein, the dissociated cells are cultured on coated plates.

Also provided herein is a screening assay comprising cultured seborrheickeratosis cells obtained using the methods described herein.

Also provided herein is a method for screening a candidate agent forinducing apoptosis, the method comprising: (a) contacting a seborrheickeratosis cell or population of seborrheic keratosis cells with acandidate agent, and (b) measuring apoptosis in the cell or populationof cells, wherein an increase in apoptosis in the cell or population ofcells indicates that the candidate agent induces apoptosis.

In one embodiment of this aspect and all other aspects described herein,the candidate agent comprises an Akt signaling pathway inhibitor.

In another embodiment of this aspect and all other aspects describedherein, the seborrheic keratosis cell or population of seborrheickeratosis cells are cultured using the method of described herein.

In another embodiment of this aspect and all other aspects describedherein, apoptosis is measured using sulforhodamine B (SRB) assay, MTTtetrazolium dye, TUNEL staining, Annexin V staining, propidium iodidestaining, DNA laddering, PARP cleavage, caspase activation, and/orassessment of cellular and nuclear morphology.

In another embodiment of this aspect and all other aspects describedherein, the candidate agent is a small molecule, a peptide inhibitor, oran RNAi molecule.

Also provided herein is an assay comprising: (a) contacting a populationof dissociated seborrheic keratosis cells with a candidate agent, (b)contacting the cells of step (a) with a detectable antibody specific foran apoptotic protein, (c) measuring the intensity of the signal from thebound, detectable antibody, (d) comparing the measured intensity of thesignal with a reference value and if the measured intensity is increasedrelative to the reference value, (e) identifying the candidate agent asan inducer of apoptosis in the cell.

In one embodiment of this aspect and all other aspects described herein,the candidate agent comprises an Akt signaling pathway inhibitor (e.g.,an Akt-1 and/or Akt-2 inhibitor and/or a pan-Akt inhibitor).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A and FIG. 1B show data from primary cultures of seborrheickeratosis (SK) cells. (FIG. 1A) Lesions were removed by curettage andwere placed in keratinocyte growth medium (INVITROGEN); part of thesamples were processed for RNA isolation for direct sequencing or fordetection of FOXN1 mRNA levels as shown in (FIG. 1B). tissues werephysically dissociated first, incubated with dispase for 24 hrs andtrypsinized for 3 min.

FIG. 2A, FIG. 2B, and FIG. 2C show data relating to the screening forsmall molecule inducers of cell death in primary SK cells. (FIG. 2A) 20small molecule inhibitors (500 nM) of kinases within the MAPK-Raf andPIK3/Akt pathway were used to treat primary SK cells (in 12 well plates)harboring either FGFR3 or/and PIK3CA mutations or none. Cell death wasdetected 24 hrs post treatment using SRB assay. (FIG. 2B) Representativeimages of cells after compound treatment. (FIG. 2C) Same molecules wereused on normal human keratinocytes in two different concentrations.

FIG. 3A, FIG. 3B, and FIG. 3C show data relating to smallmolecule-mediated inhibition of Akt signaling in primary SK cells. (FIG.3A) SK and normal keratinocytes were treated with C8 for 24 hrs and SRBstained. (FIG. 3B) and (FIG. 3C). SK cells were treated with C8 for 4hrs and subjected to Western blotting for corresponding proteins.

FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D show data relating to inhibitionof Akt signaling and induction of cell death in primary SK cells. (FIG.4A) SK cells were treated with two different Akt inhibitors and celldeath was measured 24 hrs post treatment using SRB assay. (FIG. 4B) Aktinhibitors were profiled against the human kinome and shared targetswere identified. (FIG. 4C) Cell death in SK cells after PKC inhibition.(FIG. 4D) cell death was measured in SK cells 72 hrs after transfectionwith control RNAi or RNAi for Akt1 and/or 2.

FIG. 5 shows that topical treatment of human SK explants induces tissuedestruction and cell death. Human SK explants were grown on filterinserts in contact with air and topically treated with DMSO or 1 mM C8for 48 hrs, H&E staining (left) and labeling for activated caspase 3indicate induction of cell death in compound treated explants.

FIG. 6A, FIG. 6B, and FIG. 6C show that inhibition of Akt inducesapoptosis in SK cells. (FIG. 6A) SK cells were treated with the Aktinhibitor either for 24 hrs to measure levels of cleaved PARP by Westernblotting or (FIG. 6B) for 48 hrs to detect TUNEL staining. (FIG. 6C) 24hrs post treatment pro-apoptotic Akt targets were analyzed by WB.

FIG. 7A, FIG. 7B, and FIG. 7C show that FoxO3 and p53 mediate Cell deathin SK cells upon Akt inhibition. (FIG. 7A) Cells were transfected withcontrol or FoxO3 or p53 siRNA and 48 hrs later were treated with C8.Cell death was measured 48 hrs after treatment using SRB assay. Cellsare imaged in (FIG. 7B) and protein levels were analyzed by WB in (FIG.7C).

FIG. 8 shows profiling of human SK samples: Single SKs and matchednormal skin samples were removed by curettage from 10 different patientsand subjected to Western blot analysis for levels of activated Akt andp53. The top row of arrows indicate increase of Akt phosphorylated atS473 and the bottom row of arrows show decrease of p53 levels. Part ofthe SK tissue was processed for direct PCR based sequencing formutations in FGFR3 and/or PIK3CA.

FIG. 9A and FIG. 9B show cell death in primary SK cells upon Aktinhibition (FIG. 9A) correlates with reduction of phospho GSK-3β levelsas measured by Western blot analysis (FIG. 9B).

FIG. 10 shows that elipticine and kaempferol block Akt signaling in SKcells and also induce cell death in SK cells within 48 hours.

FIG. 11 shows that pyromycin is an inducer of cell death in SK cells.

FIG. 12 shows that the p53 activator Nutlin-3 induces cells death in SKcells.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are methods and assays for isolating and culturingseborrheic keratosis cells ex vivo, a technique that permits study ofthese cells in culture. Also provided herein are screening assays usingcultured seborrheic keratosis cells and methods for treating seborrheickeratosis in a subject.

Definitions

For convenience, certain terms employed in the entire application(including the specification, examples, and appended claims) arecollected here. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs.

In connection with contacting a cell with an inhibitor of the Aktsignaling pathway, “inducing apoptosis” or “increasing cell death” in acell indicates that cell death via the apoptotic pathway in a populationof cells is at least 5% higher in populations treated with an inhibitorof the Akt signaling pathway, than in a comparable, control population,wherein no Akt signaling pathway inhibitor is present. It is preferredthat the percentage of cell death in an Akt signaling pathway inhibitortreated population is at least 10% higher, at least 20% higher, at least30% higher, at least 40% higher, at least 50% higher, at least 60%higher, at least 70% higher, at least 80% higher, at least 90% higher,at least 1-fold higher, at least 2-fold higher, at least 5-fold higher,at least 10 fold higher, at least 100 fold higher, at least 1000-foldhigher, or more than a control treated population of comparable size andculture conditions. The term “control treated population” is used hereinto describe a population of cells that has been treated with identicalmedia, viral induction, nucleic acid sequences, temperature, confluency,flask size, pH, etc., with the exception of the addition of the Aktsignaling pathway inhibitor.

By “apoptosis” is meant a cell death pathway wherein a dying celldisplays a set of well-characterized biochemical hallmarks that includecytolemmal membrane blebbing, cell soma shrinkage, chromatincondensation, nuclear disintegration, and DNA laddering. There are manywell-known assays for determining the apoptotic state of a cell,including, and not limited to: reduction of MTT tetrazolium dye, TUNELstaining, Annexin V staining, propidium iodide staining, DNA laddering,PARP cleavage, caspase activation, and assessment of cellular andnuclear morphology. Any of these or other known assays may be used inthe methods of the invention to determine whether a cell is undergoingapoptosis.

An “inhibitor” of the Akt signaling pathway, as the term is used herein,can function in a competitive or non-competitive manner, and canfunction, in one embodiment, by interfering with the expression of theAkt protein (e.g., Akt-1 and/or Akt-2) and/or a downstream protein inthe Akt pathway (e.g., FoxO3, GSK3, MDM2, etc.). Any of a number ofdifferent approaches can be taken to inhibit Akt protein expression oractivity. An Akt pathway inhibitor includes any chemical or biologicalentity that, upon treatment of a cell, results in inhibition of thebiological activity caused by activation of Akt in response to cellularsignals. Akt pathway inhibitors, include, but are not limited to, smallmolecules, antibodies or antigen-binding antibody fragments,intrabodies, aptamers, antisense constructs, RNA interference agents,and ribozymes.

As used herein, the term “candidate agent” refers to a compositionanticipated to reduce at least one symptom of a seborrheic keratosis byat least 10%, for example, a candidate agent may inhibit signalingthrough the Akt pathway or may otherwise reduce the size or appearanceof a seborrheic keratosis growth. Candidate agents can then be testedusing the screening assays described herein using primary seborrheickeratosis cells to determine if the candidate agent can reproduciblycause a desired outcome and thereby be useful as an inhibitor of the Aktsignaling pathway or a treatment for seborrheic keratoses in a subject.

A “nucleic acid”, as described herein, can be RNA or DNA, and can besingle or double stranded, and can be selected, for example, from agroup including: nucleic acid encoding a protein of interest,oligonucleotides, nucleic acid analogues, for example peptide-nucleicacid (PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acid (LNA)etc. Such nucleic acid sequences include, for example, but are notlimited to, nucleic acid sequence encoding proteins, for example thatact as transcriptional repressors, antisense molecules, ribozymes, smallinhibitory nucleic acid sequences, for example but are not limited toRNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc.

The term “therapeutically effective amount”, as used herein, refers tothe amount that is safe and sufficient to treat, lesson the appearanceof, or delay the development of a seborrheic keratosis. The amount canthus cure or result in amelioration of the symptoms of the seborrheickeratosis, slow the course of seborrheic keratosis growth orprogression, and/or slow or inhibit a symptom of a seborrheic keratosis.The effective amount for the treatment of the seborrheic keratosisdepends on the type of seborrheic keratosis to be treated, the severityof the symptoms, the subject being treated, the age and generalcondition of the subject, the mode of administration and so forth. Thus,it is not possible or prudent to specify an exact “effective amount”.However, for any given case, an appropriate “effective amount” can bedetermined by one of ordinary skill in the art using only routineexperimentation.

The term “subject” as used herein includes, without limitation, a human,mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee,baboon, or rhesus. In one embodiment, the subject is a mammal. Inanother embodiment, the subject is a human.

The term “mammal” is intended to encompass a singular “mammal” andplural “mammals,” and includes, but is not limited to humans; primatessuch as apes, monkeys, orangutans, and chimpanzees; canids such as dogsand wolves; felids such as cats, lions, and tigers; equids such ashorses, donkeys, and zebras; food animals such as cows, pigs, and sheep;ungulates such as deer and giraffes; rodents such as mice, rats,hamsters and guinea pigs; and bears. In some preferred embodiments, amammal is a human.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional nucleic acid segments canbe ligated. Another type of vector is a viral vector, wherein additionalnucleic acid segments can be ligated into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors having a bacterial originof replication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “recombinant expression vectors”,or more simply “expression vectors.” In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” can be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, lentiviruses, adenoviruses and adeno-associated viruses),which serve equivalent functions. In one embodiment, lentiviruses areused to deliver one or more siRNA molecule of the present invention to acell.

Within an expression vector, “operably linked” is intended to mean thatthe nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner which allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in atarget cell when the vector is introduced into the target cell). Theterm “regulatory sequence” is intended to include promoters, enhancersand other expression control elements (e.g., polyadenylation signals).Such regulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcell and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences).Furthermore, the RNA interfering agents may be delivered by way of avector comprising a regulatory sequence to direct synthesis of thesiRNAs of the invention at specific intervals, or over a specific timeperiod. It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the target cell, the level of expression of siRNA desired, and thelike.

The expression vectors of the invention can be introduced into targetcells to thereby produce siRNA molecules of the present invention. Inone embodiment, a DNA template, e.g., a DNA template encoding the siRNAmolecule directed against the mutant allele, may be ligated into anexpression vector under the control of RNA polymerase III (Pol III), anddelivered to a target cell. Pol III directs the synthesis of small,noncoding transcripts which 3′ ends are defined by termination within astretch of 4-5 thymidines. Accordingly, DNA templates may be used tosynthesize, in vivo, both sense and antisense strands of siRNAs whicheffect RNAi (Sui, et al. (2002) PNAS 99(8):5515).

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth. It is understood that theforegoing detailed description and the following examples areillustrative only and are not to be taken as limitations upon the scopeof the invention. Various changes and modifications to the disclosedembodiments, which will be apparent to those of skill in the art, may bemade without departing from the spirit and scope of the presentinvention. Further, all patents, patent applications, and publicationsidentified are expressly incorporated herein by reference for thepurpose of describing and disclosing, for example, the methodologiesdescribed in such publications that might be used in connection with thepresent invention. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents are based on theinformation available to the applicants and do not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

Diagnosing Seborrheic Keratosis

Seborrheic keratoses (SKs) are the most common benign epithelial tumorsin humans. The etiology of SKs are unknown but they exhibit histologicevidence of increased proliferation of keratinocytes. These lesions havean increased rate of apoptosis and several studies show that theirincidence increases with age. Some studies have found that 88% ofindividuals over age 64 have at least one SK.

SKs are characterized as a dull hyperkeratotic macule that evolves to apapulonodular lesion. They can appear as pale brown, pink, tan or brownin color and the surface can become warty or verrucous. The size variesfrom 5 mm to several centimeters and a classic “stuck on” appearance isobserved. SKs never progress to malignant tumors.

SKs commonly harbor multiple oncogenic mutations in FGFR3, PIK3CA, KRAS,HRAS, EGFR, and AKT1 oncogenes but not in tumor suppressor genes p53,TSC1, and PTEN. There is no evidence indicating that a senescenceprogram is activated in SKs. The expression profile of SKS is verysimilar to malignant skin tumors such as squamous cell carcinomas withthe exception that SKs harbor a strong activation of apro-differentiation program governed by a feedback loop betweenactivated receptor tyrosine kinase signaling (such as FGFR3) and thetranscription factor FOXN1.

Seborrheic keratoses can be easily diagnosed visually by one of skill inthe art of medicine, particularly dermatology. A seborrheic keratosis istypically a brown, black or pale growth found on the back, shoulders,face or chest. In general, a seborrheic keratosis is slightly elevatedabove the skin surface and can appear waxy or scaly. In some cases, aseborrheic keratosis can resemble a wart, an actinic keratosis, or skincancer. If a doctor suspects skin cancer, a biopsy can be performed toconfirm that a growth is a seborrheic keratosis. Such methods areroutine to those of skill in the art.

Seborrheic Keratosis and Akt Signaling

Mutations detected in SKs are observed in malignant tumors and moreimportantly, when expressed in cells or transgenic animals aresufficient to induce transformation and cause cancer. Similarly, theinventors' attempt to identify important, specific moleculardeterminants of SKs resulted in the detection of increased expression ofgrowth factors and other genes thought to be involved in epithelialtumor formation or keratinocyte differentiation⁸. The observation thatSKs rarely, if ever, become malignant is therefore perplexing. Ananalogous but yet significantly different situation occurs in benignmelanocytic nevi, which often have activating mutations in the signalingmolecule b-Raf, the same mutation that is seen in many malignantmelanomas⁹. Yet, melanocytic nevi still have a risk of malignanttransformation. Therefore SKs represent a unique opportunity to studythe genomic aberrations that are tolerated in benign tumors andinvestigate how molecular signaling driven by oncogenes function innon-transformed cells.

The most frequently mutated genes in SKs are FGFR3 and the p110α subunitof PI3K (PIK3CA), in which hotspot mutations result in constitutiveactivation and signaling through the PI3K-Akt pathway⁷. At the molecularlevel, PI3K signaling upstream of Akt (a serine-threonine kinasedownstream of PI3K, also known as PKB) activation, is negativelyregulated by the tumor suppressor PTEN¹⁰. Activated Akt signaling iscritical in promoting cell survival downstream of growth factors,oncogenes and cell stress¹¹. Akt enhances the survival of cells byblocking the function of pro-apoptotic pathways such as the p53-MDM2pathways as well as the FOXO mediated pro-apoptotic cascade¹².

Akt is a serine-threonine protein kinase that is regulated byphosphatidylinositol 3,4,5-triphosphate (PIP3) and has been implicatedin signaling of survival in a wide variety of cells, includingfibroblastic, epithelial, and neuronal cells (Franke et al. Cell 1997;1; 88:435-7; Hemmings et al. Science 1997; 275:628-30). Akt was firstrecognized as an anti-apoptotic factor during analysis of signaling byinsulin-like growth factor-1 (IGF-1), which promotes the survival ofcerebellar neurons (Dudek et al. Science 1997; 275:661-5). IGF-1 wasshown to activate PI3-kinase-triggered activation of theserine-threonine kinase, Akt.

Further definition and details of the PI3 kinase/Akt signaling pathwayare disclosed in the art e.g., Downward, J. Curr. Opin. Cell Biol. 10,262-267 (1988); Jimenez, C. et al., J. Biol. Chem. 277(44):41556-41562(2002); Kitamura, T. et al., Mol. Cell Biol. 19, 6286-6296 (1999);Ruggero D. and Sonenberg N. Oncogene. 24, 7426-34 (2005); Testa J. R.and Tsichlis P. N. Oncogene. 7391-7393 (2005); and Zhou X. M. et al., J.Biol. Chem. 275, 25046-25051 (2000).

Apoptosis

Apoptosis is a mechanism for programmed cell death that typically occursduring embryogenesis, development and during the normal physiologicalresponse to aging. Apoptosis can also be triggered in response to a cellstressor, such as heat, radiation, nutrient deprivation, viralinfection, hypoxia, increased intracellular calcium concentration and inresponse to certain glucocorticoid receptor activation. Apoptosis, inpart, initiates activation of one or more caspase signaling pathways.Caspases are strong proteases that cleave after aspartic acid residuesand once activated, are responsible for proteolytic cleavage of a broadspectrum of cellular targets that ultimately lead to cell death.Defective apoptosis regulation can lead to a variety of disorders. Forexample, impaired apoptotic activity can lead to inappropriate cellsurvival, and is associated with tumor growth, cancer, autoimmunedisease, and inflammatory disease. Conversely, pathologically highlevels of apoptosis can result in abnormal initiation of cell deathpathways, as observed in e.g., neurodegenerative diseases (e.g.,Parkinson's disease, Alzheimer's disease, dementia, and cerebralischemia, among others) and infection (e.g., AIDS).

Proteins involved in the regulation of apoptosis include, for example,caspase-3, caspase-6, caspase-9, and PARP, among others. Such proteinscan be used in immunoassays to detect apoptosis in a cell or tissue.

Other exemplary assays for determining the presence and/or the level ofapoptosis in a sample include, but are not limited to, apoptosis ismeasured using sulforhodamine B (SRB) assay, MTT tetrazolium dye, TUNELstaining, Annexin V staining, propidium iodide staining, DNA laddering,PARP cleavage, caspase activation, and/or assessment of cellular andnuclear morphology.

Inhibition of Akt

By “inhibits Akt-1 and/or Akt-2 expression” is meant that the amount ofexpression of Akt-1 and/or Akt-2 is at least 5% lower in populationstreated with an Akt signaling pathway inhibitor, than a comparable,control population, wherein no such inhibitor inhibitor is present. Itis preferred that the percentage of Akt expression (e.g., Akt-1 and/orAkt-2) in an inhibitor treated population is at least 10% lower, atleast 20% lower, at least 30% lower, at least 40% lower, at least 50%lower, at least 60% lower, at least 70% lower, at least 80% lower, atleast 90% lower, at least 1-fold lower, at least 2-fold lower, at least5-fold lower, at least 10 fold lower, at least 100 fold lower, at least1000-fold lower, or more than a comparable control treated population inwhich no inhibitor is added.

By “inhibits Akt activity” is meant that the amount of functionalactivity of Akt-1 and/or Akt-2 is at least 5% lower in populationstreated with an Akt or Akt signaling pathway inhibitor, than acomparable, control population, wherein no such inhibitor is present. Itis preferred that the percentage of Akt activity (e.g., Akt-1 and/orAkt-2 activity) in an inhibitor treated population is at least 10%lower, at least 20% lower, at least 30% lower, at least 40% lower, atleast 50% lower, at least 60% lower, at least 70% lower, at least 80%lower, at least 90% lower, at least 1-fold lower, at least 2-fold lower,at least 5-fold lower, at least 10 fold lower, at least 100 fold lower,at least 1000-fold lower, or more than a comparable control treatedpopulation in which no Akt or Akt signaling pathway inhibitor is added.At a minimum, Akt activity can be assayed by determining the amount ofAkt expression at the protein or mRNA levels, using techniques standardin the art. Alternatively, or in addition, Akt activity can bedetermined using a reporter construct, wherein the reporter construct issensitive to Akt activity.

In one embodiment, the inhibitor of Akt activity is selected from thegroup consisting of an antibody against Akt-1 and/or Akt-2 (including anantibody that acts as a pan-Akt inhibitor) or an antigen-bindingfragment thereof, a small molecule, and a nucleic acid. In oneembodiment, the nucleic acid is an Akt-1 and/or Akt-2 specific RNAinterference agent, a vector encoding the RNA interference agent, or anaptamer that binds Akt-1 and/or Akt-2.

In one embodiment, the inhibitor of Akt activity interferes with Aktinteractions with its downstream mediators. In one embodiment, thedownstream mediators are GSK3, FoxO3, MDM2, among others.

Antibody Inhibitors of the Akt Signaling Pathway

Antibodies that specifically bind Akt or an Akt downstream mediator canbe used to inhibit the Akt signaling pathway in vivo, in vitro, or exvivo. Antibodies to Akt are commercially available and/or can be raisedby one of skill in the art using well known methods. The Akt inhibitoryactivity of a given antibody, or, for that matter, any Akt inhibitor,can be assessed using methods known in the art or described herein—toavoid doubt, an antibody that inhibits Akt will cause an increase incell death. Antibody inhibitors of Akt can include polyclonal andmonoclonal antibodies and antigen-binding derivatives or fragmentsthereof. Well known antigen binding fragments include, for example,single domain antibodies (dAbs; which consist essentially of single VLor VH antibody domains), Fv fragment, including single chain Fv fragment(scFv), Fab fragment, and F(ab′)2 fragment. Methods for the constructionof such antibody molecules are well known in the art.

An “antibody” that can be used according to the methods described hereinincludes complete immunoglobulins, antigen binding fragments ofimmunoglobulins, as well as antigen binding proteins that compriseantigen binding domains of immunoglobulins. Antigen binding fragments ofimmunoglobulins include, for example, Fab, Fab′, F(ab′)2, scFv and dAbs.Modified antibody formats have been developed which retain bindingspecificity, but have other characteristics that may be desirable,including for example, bispecificity, multivalence (more than twobinding sites), and compact size (e.g., binding domains alone). Singlechain antibodies lack some or all of the constant domains of the wholeantibodies from which they are derived. Therefore, they can overcomesome of the problems associated with the use of whole antibodies. Forexample, single-chain antibodies tend to be free of certain undesiredinteractions between heavy-chain constant regions and other biologicalmolecules. Additionally, single-chain antibodies are considerablysmaller than whole antibodies and can have greater permeability thanwhole antibodies, allowing single-chain antibodies to localize and bindto target antigen-binding sites more efficiently. Furthermore, therelatively small size of single-chain antibodies makes them less likelyto provoke an unwanted immune response in a recipient than wholeantibodies. Multiple single chain antibodies, each single chain havingone VH and one VL domain covalently linked by a first peptide linker,can be covalently linked by at least one or more peptide linker to formmultivalent single chain antibodies, which can be monospecific ormultispecific. Each chain of a multivalent single chain antibodyincludes a variable light chain fragment and a variable heavy chainfragment, and is linked by a peptide linker to at least one other chain.The peptide linker is composed of at least fifteen amino acid residues.The maximum number of linker amino acid residues is approximately onehundred. Two single chain antibodies can be combined to form a diabody,also known as a bivalent dimer. Diabodies have two chains and twobinding sites, and can be monospecific or bispecific. Each chain of thediabody includes a VH domain connected to a VL domain. The domains areconnected with linkers that are short enough to prevent pairing betweendomains on the same chain, thus driving the pairing betweencomplementary domains on different chains to recreate the twoantigen-binding sites. Three single chain antibodies can be combined toform triabodies, also known as trivalent trimers. Triabodies areconstructed with the amino acid terminus of a VL or VH domain directlyfused to the carboxyl terminus of a VL or VH domain, i.e., without anylinker sequence. The triabody has three Fv heads with the polypeptidesarranged in a cyclic, head-to-tail fashion. A possible conformation ofthe triabody is planar with the three binding sites located in a planeat an angle of 120 degrees from one another. Triabodies can bemonospecific, bispecific or trispecific. Thus, antibodies useful in themethods described herein include, but are not limited to, naturallyoccurring antibodies, bivalent fragments such as (Fab′)2, monovalentfragments such as Fab, single chain antibodies, single chain Fv (scFv),single domain antibodies, multivalent single chain antibodies,diabodies, triabodies, and the like that bind specifically with anantigen.

Antibodies can also be raised against a polypeptide or portion of apolypeptide by methods known to those skilled in the art. Antibodies arereadily raised in animals such as rabbits or mice by immunization withthe gene product, or a fragment thereof. Immunized mice are particularlyuseful for providing sources of B cells for the manufacture ofhybridomas, which in turn are cultured to produce large quantities ofmonoclonal antibodies. Antibody manufacture methods are described indetail, for example, in Harlow et al., 1988. While both polyclonal andmonoclonal antibodies can be used in the methods described herein, it ispreferred that a monoclonal antibody is used where conditions requireincreased specificity for a particular protein.

Nucleic Acid Inhibitors of Akt Expression

A powerful approach for inhibiting the expression of selected targetpolypeptides is through the use of RNA interference agents. RNAinterference (RNAi) uses small interfering RNA (siRNA) duplexes thattarget the messenger RNA encoding the target polypeptide for selectivedegradation. siRNA-dependent post-transcriptional silencing of geneexpression involves cleaving the target messenger RNA molecule at a siteguided by the siRNA. “RNA interference (RNAi)” is an evolutionallyconserved process whereby the expression or introduction of RNA of asequence that is identical or highly similar to a target gene results inthe sequence specific degradation or specific post-transcriptional genesilencing (PTGS) of messenger RNA (mRNA) transcribed from that targetedgene (see Coburn, G. and Cullen, B. (2002) J. of Virology 76(18):9225),thereby inhibiting expression of the target gene. In one embodiment, theRNA is double stranded RNA (dsRNA). This process has been described inplants, invertebrates, and mammalian cells. In nature, RNAi is initiatedby the dsRNA-specific endonuclease Dicer, which promotes processivecleavage of long dsRNA into double-stranded fragments termed siRNAs.siRNAs are incorporated into a protein complex (termed “RNA inducedsilencing complex,” or “RISC”) that recognizes and cleaves target mRNAs.RNAi can also be initiated by introducing nucleic acid molecules, e.g.,synthetic siRNAs or RNA interfering agents, to inhibit or silence theexpression of target genes. As used herein, “inhibition of target geneexpression” includes any decrease in expression or protein activity orlevel of the target gene or protein encoded by the target gene ascompared to a situation wherein no RNA interference has been induced.The decrease will be of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95% or 99% or more as compared to the expression of a target geneor the activity or level of the protein encoded by a target gene whichhas not been targeted by an RNA interfering agent.

The terms “RNA interference agent” and “RNA interference” as they areused herein are intended to encompass those forms of gene silencingmediated by double-stranded RNA, regardless of whether the RNAinterfering agent comprises an siRNA, miRNA, shRNA or otherdouble-stranded RNA molecule. “Short interfering RNA” (siRNA), alsoreferred to herein as “small interfering RNA” is defined as an RNA agentwhich functions to inhibit expression of a target gene, e.g., by RNAi.An siRNA may be chemically synthesized, may be produced by in vitrotranscription, or may be produced within a host cell. In one embodiment,siRNA is a double stranded RNA (dsRNA) molecule of about 15 to about 40nucleotides in length, preferably about 15 to about 28 nucleotides, morepreferably about 19 to about 25 nucleotides in length, and morepreferably about 19, 20, 21, 22, or 23 nucleotides in length, and maycontain a 3′ and/or 5′ overhang on each strand having a length of about0, 1, 2, 3, 4, or 5 nucleotides. The length of the overhang isindependent between the two strands, i.e., the length of the overhang onone strand is not dependent on the length of the overhang on the secondstrand. Preferably the siRNA is capable of promoting RNA interferencethrough degradation or specific post-transcriptional gene silencing(PTGS) of the target messenger RNA (mRNA).

siRNAs also include small hairpin (also called stem loop) RNAs (shRNAs).In one embodiment, these shRNAs are composed of a short (e.g., about 19to about 25 nucleotide) antisense strand, followed by a nucleotide loopof about 5 to about 9 nucleotides, and the analogous sense strand.Alternatively, the sense strand may precede the nucleotide loopstructure and the antisense strand may follow. These shRNAs may becontained in plasmids, retroviruses, and lentiviruses and expressedfrom, for example, the pol III U6 promoter, or another promoter (see,e.g., Stewart, et al. (2003) RNA April; 9(4):493-501, incorporated byreference herein in its entirety). The target gene or sequence of theRNA interfering agent may be a cellular gene or genomic sequence, e.g.the Akt1 or Akt2 sequence. An siRNA may be substantially homologous tothe target gene or genomic sequence, or a fragment thereof. As used inthis context, the term “homologous” is defined as being substantiallyidentical, sufficiently complementary, or similar to the target mRNA, ora fragment thereof, to effect RNA interference of the target. Inaddition to native RNA molecules, RNA suitable for inhibiting orinterfering with the expression of a target sequence include RNAderivatives and analogs. Preferably, the siRNA is identical to itstarget. The siRNA preferably targets only one sequence. Each of the RNAinterfering agents, such as siRNAs, can be screened for potentialoff-target effects by, for example, expression profiling. Such methodsare known to one skilled in the art and are described, for example, inJackson et al. Nature Biotechnology 6:635-637, 2003. In addition toexpression profiling, one may also screen the potential target sequencesfor similar sequences in the sequence databases to identify potentialsequences which may have off-target effects. For example, according toJackson et al. (Id.), 15, or perhaps as few as 11 contiguousnucleotides, of sequence identity are sufficient to direct silencing ofnon-targeted transcripts. Therefore, one may initially screen theproposed siRNAs to avoid potential off-target silencing using thesequence identity analysis by any known sequence comparison methods,such as BLAST. siRNA sequences are chosen to maximize the uptake of theantisense (guide) strand of the siRNA into RISC and thereby maximize theability of RISC to target human GGT mRNA for degradation. This can beaccomplished by scanning for sequences that have the lowest free energyof binding at the 5′-terminus of the antisense strand. The lower freeenergy leads to an enhancement of the unwinding of the 5′-end of theantisense strand of the siRNA duplex, thereby ensuring that theantisense strand will be taken up by RISC and direct thesequence-specific cleavage of the human Akt1 or Akt2 mRNA. siRNAmolecules need not be limited to those molecules containing only RNA,but, for example, further encompasses chemically modified nucleotidesand non-nucleotides, and also include molecules wherein a ribose sugarmolecule is substituted for another sugar molecule or a molecule whichperforms a similar function. Moreover, a non-natural linkage betweennucleotide residues can be used, such as a phosphorothioate linkage. TheRNA strand can be derivatized with a reactive functional group of areporter group, such as a fluorophore. Particularly useful derivativesare modified at a terminus or termini of an RNA strand, typically the 3′terminus of the sense strand. For example, the 2′-hydroxyl at the 3′terminus can be readily and selectively derivatizes with a variety ofgroups. Other useful RNA derivatives incorporate nucleotides havingmodified carbohydrate moieties, such as 2′O-alkylated residues or2′-O-methyl ribosyl derivatives and 2′-O-fluoro ribosyl derivatives. TheRNA bases may also be modified. Any modified base useful for inhibitingor interfering with the expression of a target sequence may be used. Forexample, halogenated bases, such as 5-bromouracil and 5-iodouracil canbe incorporated. The bases may also be alkylated, for example,7-methylguanosine can be incorporated in place of a guanosine residue.Non-natural bases that yield successful inhibition can also beincorporated. The most preferred siRNA modifications include2′-deoxy-2′-fluorouridine or locked nucleic acid (LAN) nucleotides andRNA duplexes containing either phosphodiester or varying numbers ofphosphorothioate linkages. Such modifications are known to one skilledin the art and are described, for example, in Braasch et al.,Biochemistry, 42: 7967-7975, 2003. Most of the useful modifications tothe siRNA molecules can be introduced using chemistries established forantisense oligonucleotide technology. Preferably, the modificationsinvolve minimal 2′-O-methyl modification, preferably excluding suchmodification. Modifications also preferably exclude modifications of thefree 5′-hydroxyl groups of the siRNA.

In a preferred embodiment, the RNA interference agent is delivered oradministered in a pharmaceutically acceptable carrier. Additionalcarrier agents, such as liposomes, can be added to the pharmaceuticallyacceptable carrier. In another embodiment, the RNA interference agent isdelivered by a vector encoding small hairpin RNA (shRNA) in apharmaceutically acceptable carrier to the cells in an organ of anindividual. The shRNA is converted by the cells after transcription intosiRNA capable of targeting, for example, Ak-t1 or Akt-2.

In one embodiment, the vector is a regulatable vector, such astetracycline inducible vector. Methods described, for example, in Wanget al. Proc. Natl. Acad. Sci. 100: 5103-5106, using pTet-On vectors (BDBiosciences Clontech, Palo Alto, Calif.) can be used. In one embodiment,the RNA interference agents used in the methods described herein aretaken up actively by cells in vivo following intravenous injection,e.g., hydrodynamic injection, without the use of a vector, illustratingefficient in vivo delivery of the RNA interfering agents. One method todeliver the siRNAs is catheterization of the blood supply vessel of thetarget organ. Other strategies for delivery of the RNA interferenceagents, e.g., the siRNAs or shRNAs used in the methods of the invention,may also be employed, such as, for example, delivery by a vector, e.g.,a plasmid or viral vector, e.g., a lentiviral vector. Such vectors canbe used as described, for example, in Xiao-Feng Qin et al. Proc. Natl.Acad. Sci. U.S.A., 100: 183-188. Other delivery methods include deliveryof the RNA interfering agents, e.g., the siRNAs or shRNAs of theinvention, using a basic peptide by conjugating or mixing the RNAinterfering agent with a basic peptide, e.g., a fragment of a TATpeptide, mixing with cationic lipids or formulating into particles. TheRNA interference agents, e.g., the siRNAs targeting Akt-1 or Akt-2 mRNA,may be delivered singly, or in combination with other RNA interferenceagents, e.g., siRNAs, such as, for example siRNAs directed to othercellular genes. Akt siRNAs may also be administered in combination withother pharmaceutical agents which are used to treat or prevent diseasesor disorders associated with oxidative stress, especially respiratorydiseases, and more especially asthma. Synthetic siRNA molecules,including shRNA molecules, can be obtained using a number of techniquesknown to those of skill in the art. For example, the siRNA molecule canbe chemically synthesized or recombinantly produced using methods knownin the art, such as using appropriately protected ribonucleosidephosphoramidites and a conventional DNA/RNA synthesizer (see, e.g.,Elbashir, S. M. et al. (2001) Nature 411:494-498; Elbashir, S. M., W.Lendeckel and T. Tuschl (2001) Genes & Development 15:188-200; Harborth,J. et al. (2001) J. Cell Science 114:4557-4565; Masters, J. R. et al.(2001) Proc. Natl. Acad. Sci., USA 98:8012-8017; and Tuschl, T. et al.(1999) Genes & Development 13:3191-3197). Alternatively, severalcommercial RNA synthesis suppliers are available including, but notlimited to, Proligo (Hamburg, Germany), Dharmacon Research (Lafayette,Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, Ill.,USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass.,USA), and Cruachem (Glasgow, UK). As such, siRNA molecules are notoverly difficult to synthesize and are readily provided in a qualitysuitable for RNAi. In addition, dsRNAs can be expressed as stem loopstructures encoded by plasmid vectors, retroviruses and lentiviruses(Paddison, P. J. et al. (2002) Genes Dev. 16:948-958; McManus, M. T. etal. (2002) RNA 8:842-850; Paul, C. P. et al. (2002) Nat. Biotechnol.20:505-508; Miyagishi, M. et al. (2002) Nat. Biotechnol. 20:497-500;Sui, G. et al. (2002) Proc. Natl. Acad. Sci., USA 99:5515-5520;Brummelkamp, T. et al. (2002) Cancer Cell 2:243; Lee, N. S., et al.(2002) Nat. Biotechnol. 20:500-505; Yu, J. Y., et al. (2002) Proc. Natl.Acad. Sci., USA 99:6047-6052; Zeng, Y., et al. (2002) Mol. Cell9:1327-1333; Rubinson, D. A., et al. (2003) Nat. Genet. 33:401-406;Stewart, S. A., et al. (2003) RNA 9:493-501). These vectors generallyhave a polIII promoter upstream of the dsRNA and can express sense andantisense RNA strands separately and/or as a hairpin structures. Withincells, Dicer processes the short hairpin RNA (shRNA) into effectivesiRNA. The targeted region of the siRNA molecule of the presentinvention can be selected from a given target gene sequence, e.g., anAkt-1 or Akt-2 coding sequence, beginning from about 25 to 50nucleotides, from about 50 to 75 nucleotides, or from about 75 to 100nucleotides downstream of the start codon. Nucleotide sequences maycontain 5′ or 3′ UTRs and regions nearby the start codon. One method ofdesigning a siRNA molecule of the present invention involves identifyingthe 23 nucleotide sequence motif AA(N19)TT (SEQ. ID. NO. 21) (where Ncan be any nucleotide) and selecting hits with at least 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% G/C content. The “TT” portionof the sequence is optional. Alternatively, if no such sequence isfound, the search may be extended using the motif NA(N21), where N canbe any nucleotide. In this situation, the 3′ end of the sense siRNA maybe converted to TT to allow for the generation of a symmetric duplexwith respect to the sequence composition of the sense and antisense 3′overhangs. The antisense siRNA molecule may then be synthesized as thecomplement to nucleotide positions 1 to 21 of the 23 nucleotide sequencemotif. The use of symmetric 3′ TT overhangs may be advantageous toensure that the small interfering ribonucleoprotein particles (siRNPs)are formed with approximately equal ratios of sense and antisense targetRNA-cleaving siRNPs (Elbashir et al., (2001) supra and Elbashir et al.,2001 supra). Analysis of sequence databases, including but not limitedto the NCBI, BLAST, Derwent and GenSeq as well as commercially availableoligosynthesis companies such as Oligoengine®, may also be used toselect siRNA sequences against EST libraries to ensure that only onegene is targeted.

siRNA sequences to target Akt-1, Akt-2, Akt-3, FOXO3, and p53, amongothers, can also be obtained commercially from e.g., INVITROGEN™, THERMOSCIENTIFIC™, ORIGENE™, among others. For example, Validated StealthsiRNAs can be obtained for Akt-1 (cat. No. VHS40082), Akt-2 (cat. No.VHS41339), and Akt-3 (cat no. HSS115178) from INVITROGEN™. In addition,ON-TARGETplus SMARTpool siRNAS™ can be obtained from THERMO SCIENTIFIC™for FOXO3 (Human FOXO3A(2309); cat no. L-003007-00-0005) and for p53(Human TP53 (7157); cat. no. L-003329-00-0005).

Delivery of RNA Interfering Agents

Methods of delivering RNA interference agents, e.g., an siRNA, orvectors containing an RNA interference agent, to the target cells, e.g.,seborrheic keratosis cells, skin cells, or other desired target cells,for uptake include injection of a composition containing the RNAinterference agent, e.g., an siRNA, or directly contacting the cell,e.g., a seborrheic keratosis cell, with a composition comprising an RNAinterference agent, e.g., an siRNA. In another embodiment, RNAinterference agent, e.g., an siRNA may be injected directly into anyblood vessel, such as vein, artery, venule or arteriole, via, e.g.,hydrodynamic injection or catheterization. Administration may be by asingle injection or by two or more injections. The RNA interferenceagent is delivered in a pharmaceutically acceptable carrier. One or moreRNA interference agent may be used simultaneously. In one embodiment, asingle siRNA that targets human Akt is used. In one embodiment, specificcells are targeted with RNA interference, limiting potential sideeffects of RNA interference caused by non-specific targeting of RNAinterference. The method can use, for example, a complex or a fusionmolecule comprising a cell targeting moiety and an RNA interferencebinding moiety that is used to deliver RNA interference effectively intocells. For example, an antibody-protamine fusion protein when mixed withsiRNA, binds siRNA and selectively delivers the siRNA into cellsexpressing an antigen recognized by the antibody, resulting in silencingof gene expression only in those cells that express the antigen. ThesiRNA or RNA interference-inducing molecule binding moiety is a proteinor a nucleic acid binding domain or fragment of a protein, and thebinding moiety is fused to a portion of the targeting moiety. Thelocation of the targeting moiety can be either in the carboxyl-terminalor amino-terminal end of the construct or in the middle of the fusionprotein. A viral-mediated delivery mechanism can also be employed todeliver siRNAs to cells in vitro and in vivo as described in Xia, H. etal. (2002) Nat Biotechnol 20(10):1006). Plasmid- or viral-mediateddelivery mechanisms of shRNA may also be employed to deliver shRNAs tocells in vitro and in vivo as described in Rubinson, D. A., et al.((2003) Nat. Genet. 33:401-406) and Stewart, S. A., et al. ((2003) RNA9:493-501). The RNA interference agents, e.g., the siRNAs or shRNAs, canbe introduced along with components that perform one or more of thefollowing activities: enhance uptake of the RNA interfering agents,e.g., siRNA, by the cell, e.g., lymphocytes or other cells, inhibitannealing of single strands, stabilize single strands, or otherwisefacilitate delivery to the target cell and increase inhibition of thetarget gene, e.g., Akt-1 or Akt-2. The dose of the particular RNAinterfering agent will be in an amount necessary to effect RNAinterference, e.g., post translational gene silencing (PTGS), of theparticular target gene, thereby leading to inhibition of target geneexpression or inhibition of activity or level of the protein encoded bythe target gene.

In one embodiment, the seborrheic keratosis cell is contacted ex vivo orin vitro. In one embodiment, the composition inhibits Akt-1 and/or Akt-2expression.

Small Molecule Inhibition of the Akt Signaling Pathway

As used herein, the term “small molecule” refers to a chemical agentincluding, but not limited to, peptides, peptidomimetics, amino acids,amino acid analogs, polynucleotides, polynucleotide analogs, aptamers,nucleotides, nucleotide analogs, organic or inorganic compounds (i.e.,including heteroorganic and organometallic compounds) having a molecularweight less than about 10,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 5,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 1,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 500 grams per mole, and salts, esters,and other pharmaceutically acceptable forms of such compounds.

Essentially any small molecule inhibitor of the Akt signaling pathwaycan be used in the treatment of seborrheic keratosis using the methodsdescribed herein. In one embodiment, the small molecule inhibitor of theAkt signaling pathway is an inhibitor of Akt-1, Akt-2, Akt-3 or acombination thereof. In another embodiment, the small molecule inhibitorof the Akt signaling pathway is an ATP-competitive Akt inhibitor. Inanother embodiment, the ATP-competitive Akt inhibitor reducesphosphorylation of GSK-3β.

Some non-limiting examples of small molecule compounds useful in thetreatment of seborrheic keratosis as described herein includekaempferol, ellipticine (with Akt inhibition), nutlin-3 and puromycin.

Pharmaceutically Acceptable Carriers

As used herein, the term “pharmaceutically acceptable”, and grammaticalvariations thereof, as they refer to compositions, carriers, diluentsand reagents, are used interchangeably and represent that the materialsare capable of administration to or upon a mammal without the productionof undesirable physiological effects such as nausea, dizziness, gastricupset and the like. Each carrier must also be “acceptable” in the senseof being compatible with the other ingredients of the formulation. Apharmaceutically acceptable carrier will not promote the raising of animmune response to an agent with which it is admixed, unless so desired.The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in the artand need not be limited based on formulation. The pharmaceuticalformulation contains a compound as described herein in combination withone or more pharmaceutically acceptable ingredients. The carrier can bein the form of a solid, semi-solid or liquid diluent, cream or acapsule. Typically such compositions are prepared as injectable eitheras liquid solutions or suspensions, however, solid forms suitable forsolution, or suspensions, in liquid prior to use can also be prepared.The preparation can also be emulsified or presented as a liposomecomposition. The active ingredient can be mixed with excipients whichare pharmaceutically acceptable and compatible with the activeingredient and in amounts suitable for use in the therapeutic methodsdescribed herein. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol or the like and combinations thereof. Inaddition, if desired, the composition can contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents and the like which enhance the effectiveness of the activeingredient. The therapeutic composition of the present invention caninclude pharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.Physiologically tolerable carriers are well known in the art. Exemplaryliquid carriers are sterile aqueous solutions that contain no materialsin addition to the active ingredients and water, or contain a buffersuch as sodium phosphate at physiological pH value, physiological salineor both, such as phosphate-buffered saline. Still further, aqueouscarriers can contain more than one buffer salt, as well as salts such assodium and potassium chlorides, dextrose, polyethylene glycol and othersolutes. Liquid compositions can also contain liquid phases in additionto and to the exclusion of water. Exemplary of such additional liquidphases are glycerin, vegetable oils such as cottonseed oil, andwater-oil emulsions. The amount of an active agent used in the inventionthat will be effective in the treatment of a particular disorder orcondition will depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques. The phrase“pharmaceutically acceptable carrier or diluent” means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject agents fromone organ, or portion of the body, to another organ, or portion of thebody.

Dosage, Administration and Efficacy

As used herein, “administered” refers to the placement of an agent thatinduces apoptosis (e.g., an inhibitor of the Akt signaling pathway) intoa subject by a method or route which results in at least partiallocalization of the inhibitor at a desired site. An agent which inducesapoptosis can be administered by any appropriate route which results ineffective treatment in the subject, i.e. administration results indelivery to a desired location in the subject where at least a portionof the composition delivered, i.e. at least one agent which inhibitsAkt, is active in the desired site for a period of time. The period oftime the inhibitor is active depends on the half life in vivo afteradministration to a subject, and can be as short as a few hours, e. g.twenty-four hours, to a few days, to as long as several years. Modes ofadministration include injection, infusion, instillation, topical oringestion. “Injection” includes, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intraventricular,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular,subarachnoid, intraspinal, intracerebro spinal, and intrasternalinjection and infusion. In one embodiment, the mode of administration istopical.

In a method for treating seborrheic keratosis (SK), an effective amountof an agent that induces apoptosis is administered to a patientdiagnosed as having one or more SKs. In one embodiment, the subject canbe a mammal (e.g., a primate or a non-primate mammal). In anotherembodiment, the mammal can be a human, although the approach iseffective with respect to all mammals. In one embodiment, the methodcomprises administering to the primate subject (e.g., a human) aneffective amount of a pharmaceutical composition comprising an agentthat induces apoptosis. An “effective amount” means an amount or dosegenerally sufficient to bring about the desired therapeutic orprophylactic benefit in subjects undergoing treatment. Effective amountsor doses of an agent that induces apoptosis for treatment as describedherein can be ascertained by routine methods such as modeling, doseescalation studies or clinical trials, and by taking into considerationroutine factors, e.g., the mode or route of administration of agentdelivery, the pharmacokinetics of the composition, the severity andcourse of the disorder or condition, the subject's previous or ongoingtherapy, the subject's health status and response to drugs, and thejudgment of the treating physician. An exemplary dose for a human is inthe range of from about 0.001 to about 1 g of subject's body weight perday.

While the dosage range for the composition comprising an agent thatinduces apoptosis depends upon the potency of the composition, andincludes amounts large enough to produce the desired effect (e.g.,increase in cell death), the dosage should not be so large as to causeunacceptable adverse side effects. Generally, the dosage will vary withthe formulation (e.g., oral, topical, i.v. or subcutaneousformulations), and with the age, condition, and sex of the patient. Thedosage can be determined by one of skill in the art and can also beadjusted by the individual physician in the event of any complication.Typically, the dosage will range from 0.001 mg/day to 500 mg/day. Insome embodiments, the dosage range is from 0.001 mg/day to 400 mg/day,from 0.001 mg/day to 300 mg/day, from 0.001 mg/day to 200 mg/day, from0.001 mg/day to 100 mg/day, from 0.001 mg/day to 50 mg/day, from 0.001mg/day to 25 mg/day, from 0.001 mg/day to 10 mg/day, from 0.001 mg/dayto 5 mg/day, from 0.001 mg/day to 1 mg/day, from 0.001 mg/day to 0.1mg/day, from 0.001 mg/day to 0.005 mg/day. Alternatively, the dose rangewill be titrated to maintain serum levels between 5 μg/mL and 30 m/mL.Administration of the doses recited above can be repeated for a limitedperiod of time or as necessary. In some embodiments, the doses are givenor applied once a day, or multiple times a day, for example but notlimited to three times a day. In one embodiment, the doses recited aboveare administered daily for several weeks or months. The duration oftreatment depends upon the subject's clinical progress andresponsiveness to therapy. Continuous, relatively low maintenance dosesare contemplated after an initial higher therapeutic dose. Agents usefulin the methods and compositions described herein can be administeredtopically, intravenously (by bolus or continuous infusion), orally, byinhalation, intraperitoneally, intramuscularly, subcutaneously,intracavity, and can be delivered by peristaltic means, if desired, orby other means known by those skilled in the art. Although uncommon forthe treatment of an SK, the agent can be administered systemically.Therapeutic compositions containing at least one agent can beconventionally administered in a unit dose. The term “unit dose” whenused in reference to a therapeutic composition refers to physicallydiscrete units suitable as unitary dosage for the subject, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requiredphysiologically acceptable diluent, i.e., carrier, or vehicle.

The efficacy of a treatment comprising an agent that induces apoptosisin the treatment of an SK can be determined by the skilled clinician.However, a treatment is considered “effective treatment,” as the term isused herein, if any one or all of the signs or symptoms of, as but oneexample, appearance of the SK are altered in a beneficial manner, otherclinically accepted symptoms or markers of disease are improved orameliorated, e.g., by at least 10% following treatment with aninhibitor. Methods of measuring these indicators are known to those ofskill in the art and/or described herein.

References or Reference Samples

The terms “reference level,” “reference sample,” and “reference” areused interchangeably herein and refer to the level of expression oractivity of a protein in the Akt signaling pathway (e.g., Akt-1 and/orAkt-2) in a known sample against which another sample is compared (i.e.,a skin sample obtained from a subject having a seborrheic keratosis). Astandard is useful for determining the amount of, or the relativeincrease/decrease of apoptosis or cell death in a biological sample. Astandard serves as a reference level for comparison, such that samplescan be normalized to an appropriate standard in order to infer thepresence, absence or extent of apoptosis in a subject. In oneembodiment, a biological standard is obtained from the same individualthat is to be tested or treated as described herein, prior to theinitiation of treatment. Alternatively, a standard can be from the sameindividual having been taken at a time after the onset or diagnosis of aseborrheic keratosis. In such instances, the standard can provide ameasure of the efficacy of treatment. A standard level can be obtained,for example, from a known biological sample from a different individual(e.g., not the individual being tested) that is substantially free of aseborrheic keratosis. A known sample can also be obtained by poolingsamples from a plurality of individuals to produce a standard over anaveraged population, wherein a standard represents an average level ofapoptosis among a population of individuals (e.g., a population ofindividuals having seborrheic keratosis (SK)). Thus, the level ofapoptosis in a standard obtained in this manner is representative of anaverage level of cell death in a general population of individualshaving an SK. An individual sample is compared to this populationstandard by comparing the level of apoptosis from a sample relative tothe population standard. Generally, an increase in the amount of celldeath over the standard will indicate the efficacy of treatment with thecomposition, while a decrease in the amount of apoptosis will indicatethat the treatment is not effective for reducing a symptom of an SK inthat individual. It should be noted that there is often variabilityamong individuals in a population, such that some individuals will havehigher levels of apoptosis, while other individuals have lower levels.However, one skilled in the art can make logical inferences on anindividual basis regarding the detection and treatment of an SK asdescribed herein. A standard or series of standards can also besynthesized. A known amount of an apoptotic marker (or a series of knownamounts) can be prepared within the typical expression range for themarker that is observed in a general population. This method has anadvantage of being able to compare the extent of disease in one or moreindividuals in a mixed population. This method can also be useful forsubjects who lack a prior sample to act as a standard or for routinefollow-up post-diagnosis. This type of method can also allowstandardized tests to be performed among several clinics, institutions,or countries etc.

Screening Assays

Screening assays as contemplated herein can be used to identifymodulators, i.e., candidate or test compounds or agents (e.g., peptides,antibodies, peptidomimetics, small molecules (organic or inorganic) orother drugs) which modulate apoptosis or Akt signaling pathway activity.These assays are designed to identify compounds, for example, thatinduce cell death, particularly via apoptosis, e.g., a modulator of theAkt signaling pathway.

In another embodiment, an assay is a cell-based assay comprisingcontacting a seborrheic keratosis cell in culture with a candidate agentand determining the ability of the candidate agent to modulate (e.g.,induce or inhibit) apoptosis and/or Akt signaling pathway activity.

The test compounds or candidate agents can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des.12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

The methods described herein further pertain to novel agents identifiedby the above-described screening assays. With regard to intervention,any treatments which modulate apoptosis and/or activity of the Aktsignaling pathway should be considered as candidates for humantherapeutic intervention.

The present invention can be defined in any of the following numberedparagraphs:

1. A method for treating a seborrheic keratosis in a subject, the methodcomprising administering a therapeutically effective amount of acomposition that inhibits the Akt signaling pathway to a subject havinga seborrheic keratosis.

2. The method of paragraph 1, wherein the composition is appliedtopically or administered systemically.

3. The method of paragraph 1, further comprising a step of diagnosingthe subject with a seborrheic keratosis.

4. The method of paragraph 1, wherein the therapeutically effectiveamount of the composition does not substantially affect the survival ofnormal keratinocytes.

5. The method of paragraph 1, wherein the composition comprises a smallmolecule, a peptide inhibitor, or an RNAi molecule.

6. The method of paragraph 1, wherein the composition is an Akt-1 and/oran Akt-2 inhibitor.

7. The method of paragraph 1, wherein the composition further comprisesa pharmaceutically acceptable carrier.

8. A method for inducing apoptosis in a seborrheic keratosis cell, themethod comprising contacting a seborrheic keratosis cell with aneffective amount of a composition that inhibits Akt signaling, therebyinducing apoptosis in the cell.

9. The method of paragraph 8, wherein the effective amount of thecomposition does not substantially affect the survival of normalkeratinocytes.

10. The method of paragraph 8, wherein the composition comprises a smallmolecule, a peptide inhibitor, or an RNAi molecule.

11. The method of paragraph 8, wherein the composition is an Akt-1and/or an Akt-2 inhibitor.

12. A method for culturing seborrheic keratosis cells ex vivo, themethod comprising:

(a) contacting a biological sample comprising seborrheic keratosis cellsobtained from a subject with a solution comprising a dispase enzyme at atemperature and for a time sufficient to initiate dissociation of theseborrheic keratosis cells from the biological sample, and

(b) culturing the dissociated seborrheic keratosis cells.

13. The method of paragraph 12, wherein the temperature is below astandard room temperature of 21° C.

14. The method of paragraph 12, wherein the time sufficient to initiatedigestion of the seborrheic keratosis cells is at least 15 hours.

15. The method of paragraph 12, further comprising a step of contactingthe biological sample comprising seborrheic keratosis cells with anadditional protease.

16. The method of paragraph 15, wherein the additional protease isTrypsin.

17. The method of paragraph 10, further comprising a step of adding aculture medium and filtering larger particles from the dissociated cellsbefore the culturing of step (b).

18. The method of paragraph 10, wherein the dissociated cells arecultured on coated plates.

19. A screening assay comprising cultured seborrheic keratosis cellsobtained using the method of paragraph 10.

20. A method for screening a candidate agent for inducing apoptosis, themethod comprising:

(a) contacting a seborrheic keratosis cell or population of seborrheickeratosis cells with a candidate agent, and

(b) measuring apoptosis in the cell or population of cells, wherein anincrease in apoptosis in the cell or population of cells indicates thatthe candidate agent induces apoptosis.

21. The method of paragraph 20, wherein the candidate agent comprises anAkt signaling pathway inhibitor.

22. The method of paragraph 20, wherein the seborrheic keratosis cell orpopulation of seborrheic keratosis cells are cultured using the methodof paragraph 10.

23. The method of paragraph 20, wherein apoptosis is measured usingsulforhodamine B (SRB) assay, MTT tetrazolium dye, TUNEL staining,Annexin V staining, propidium iodide staining, DNA laddering, PARPcleavage, caspase activation, and/or assessment of cellular and nuclearmorphology.24. The method of paragraph 20, wherein the candidate agent is a smallmolecule, a peptide inhibitor, or an RNAi molecule.25. An assay comprising:

(a) contacting a population of dissociated seborrheic keratosis cellswith a candidate agent,

(b) contacting the cells of step (a) with a detectable antibody specificfor an apoptotic protein,

(c) measuring the intensity of the signal from the bound, detectableantibody,

(d) comparing the measured intensity of the signal with a referencevalue and if the measured intensity is increased relative to thereference value,

(e) identifying the candidate agent as an inducer of apoptosis in thecell.

26. The assay of paragraph 25, wherein the candidate agent comprises anAkt signaling pathway inhibitor.

27. The assay of paragraph 25, wherein the population of seborrheickeratosis cells is cultured using the method of paragraph 10.

28. The assay of paragraph 25, wherein the apoptotic protein is acaspase protein, a PARP protein, or a cleavage product thereof.

29. A method for culturing seborrheic keratosis cells ex vivo, themethod comprising:

(a) contacting a biological sample comprising sebborheic keratosis cellsobtained from a subject with a solution comprising initially a dispaseenzyme and subsequently a trypsin enzyme at a temperature and for a timesufficient to dissociate seborrheic keratosis cells from the biologicalsample, and

(b) culturing the dissociated seborrheic keratosis cells.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all referencescited throughout this application, as well as the figures and table areincorporated herein by reference.

EXAMPLES

Although recent reports indicate that when compared to normal skin, SKsshowed significantly elevated levels of phosphorylated Akt, very littleis known about the biological significance of these findings partiallydue to the fact that efforts to study SKs have been hampered by theinability to culture the cells from these lesions in vitro, a problemcommon to many benign tumors. The inventors' have overcome this obstacleand also developed an explant technique that permits the entire biopsiedSK specimens from patients to be studied for several days in thelaboratory. Panels of specific signaling kinase inhibitors were used tomap out the molecular pathways critical for SK cell viability.Specifically, the signaling kinase Akt is crucial to prevent SK cellsfrom undergoing programmed cell death. Both small molecule inhibitorsand Akt siRNA knockdown induce caspase-dependent cell death via FoxO3activation. Endogenous wild-type p53 also appears to be critical in bothmaintaining a benign tumor state and in directing the apoptotic programafter Akt inhibition. Based on these findings, the inventorshypothesized that genomic alteration in SK cells results in constitutiveactivation of the Akt and its downstream targets such as FoxO3A pathway,which is essential for the growth and survival of these SK lesions invitro as well as in vivo.

Currently there are no approved pharmacological treatments for SKs. Anunderstanding of, and perhaps pharmacologic control over, thisbenign-malignant switch could prove to impact treatment of SKs as wellas many other types of benign epidermal lesions.

Example 1 Primary Cultures from Patient Samples are a Novel Approach toStudy SKs

The data presented herein as well as evidence from recently publishedreports^(5, 7) point to a unique combination of oncogenic genomicaberrations in SK lesions. However the molecular consequences of thesechanges could be studied only to a limited extent at a histologicallevel in harvested tissue or through correlation with similar changes innormal cultured human keratinocytes. Therefore, the inventors sought toextend the ability to address questions and hypothesize aboutdeterminates of benign epidermal tumor development in a well-defined invitro cell culture system. This turned out to be a challenging taskrequiring months and months of frustrating attempts to reach theappropriate combination of enzymatic digestion and physical tissuedissociation. Notably SKs can be removed by a surgical technique, whichdoes not harvest full thickness tissue but aims at preservation of thedermis, thus making the regular “peal off” of the epidermis afterdispase incubation nearly impossible.

The inventors were able to modify the previously describedtechnique^(23, 24) for primary cultures of normal human keratinocytesand adapted it for successful isolation of single SK cell suspension andsubsequent expansion in culture (FIG. 3A). Three major genomic types ofSK primary cells were identified: 1) with mutations in FGFR3; 2) withmutations in PIK3CA; and 3) no detected mutations. Cells were culturedafter sequencing of the target genes for hotspot aberrations as shown inFIG. 6. Passaging up to three times was successful at this time pointbut the inventors chose to perform all the experiments with cells ofpassage one or two. Importantly, all types of primary SK cells showedoverexpression of FOXN1 in culture (FIG. 3B).

Example 2 Primary SK Cells are Dependent on Activated Akt Signaling forSurvival

Considerable evidence suggests that a hallmark of SK lesions is enhancedsurvival and lower apoptosis rates²⁵. The inventors sought to identifythe major pathways responsible for this feature. Previouslycharacterized genomic alterations in SKs point to the fact thatpathological activation of receptor tyrosine kinase signaling pathwayscan drive survival of these types of tumors. Therefore, it washypothesized that inhibition of kinase signaling would induce cell deathin SK cells.

Due to the fragility of the primary cultures, RNAi transfection in ahigh throughput fashion turned out to be challenging, which prompted theuse of a collection of specific small molecule kinase inhibitors todetermine which kinase is essential for survival of SK cells but not ofnormal human keratinocytes. This level of selectivity was introduced inorder to specifically target dependency caused by genomic alterations(both mutations and overexpression). The experiments were performedusing all three types of primary SK cells (with FGFR3 mutations; withPIK3CA mutations; without mutations in any of these genes) andsurprisingly the results were consistently similar (FIG. 4). Out of all20 kinase inhibitors tested three had a killing effect above 60% andonly one compound scored above 80% (FIG. 4A). Importantly, the inventorsdid not observe considerable cell death with these compounds in bothmatched and non-matched normal primary keratinocytes. The compounds,which scored as SK cell death inducers in this assay were modulators ofthe same signaling pathway: inhibitors of PI3K/mTOR (weaker activity:compounds 11 (C11) and 12 (C12)) and an ATP-competitive Akt inhibitor(compound 8, (C8), also known as A-443654 26), which had a very strongactivity.

Interestingly the allosteric Akt inhibitor included in the screen,compound 9 (C9, also known as AT7867) did not affect the viability ofthe SK cells. This effect was confirmed and a dose-response killingeffect of C8 in SK cells was observed, while normal keratinocytesremained unaffected even at higher doses. In order to verify theactivity of C8 in the present assay, the phosphorylation status of Aktwas studied in the SK cells and a paradoxical hyperphosphorylation ofAkt at T308 and 5473 was observed, which is characteristic for thesetype of molecules^(27, 28). As recently reported thishyperphosphorylation is due to the docking of the compound in the ATPbinding site, which prevents the Akt interaction with its downstreamtargets²⁷. As shown in FIG. 5B SK cells treated with C8 indeed have ahyperphosphorylated Akt, but importantly the Akt downstream signalingwas shut down (FIG. 5C). The sensitivity of the cells against ATPcompetitive inhibitors was confirmed again since the allostericinhibitor did not show any effects (FIG. 5A).

SK cells were treated with another ATP-competitive Akt inhibitor(GSK690693, 29) and a similar induction of cell death was observed (FIG.5A). Small molecule kinase inhibitors usually have a high degree ofspecificity towards kinases in general but often these inhibitoryeffects are shared among several kinases, although in different doses.While this is in most of the cases considered a weakness of theapproach, it can also give some evidence about other possible targetsinvolved in the process of interest. Therefore the inventors profiledthe two effective compounds for their targets (in collaboration with Dr.N. Gray, DFCI) and determined if any other kinases might be involved inthe survival of SK cells. Surprisingly, although C8 and GSK690693 affectseveral off-target kinases, only members of the Akt and PKC family wereshared targets (FIG. 6B). Therefore, a pan-PKC inhibitor was tested onthe SK cells but no killing effect was observed (FIG. 6C), indicatingthat inhibition of Akt activity was indeed essential for cell death inSK cells. In addition, the inventors were able to confirm this bydown-modulation of Akt family members using an RNAi approach. Depletionof Akt 1 and 2 through RNAi (separate or together) had a significantkilling effect on SK cells (FIG. 6)

It is now widely accepted that growth, differentiation and death ofkeratinocytes in culture differs in significant aspects from that inintact skin. Therefore, in order to closely reproduce the in vivosituation for SK tumors, the inventors utilized an ex vivo explantsystem¹⁶ for culturing freshly excised SK lesions for up to 7 days. SKexplants were treated by topical application of 1 mM of C8 for 48 hrsand stained for activated Caspase 3 to detect cell death. As shown inFIG. 7, in the ex vivo model, the inhibition of Akt activity with mosteffective AKT inhibitor C8 resulted in a significant cell death of SKcells.

Therefore, the data described herein indicate that SK cells depend onthe constitutive activation of the Akt pathway for survival, thus smallmolecule or RNAi based inhibition of the kinase is able to induce rapidcell death in these benign tumors.

In addition, ATP-competitive Akt inhibitors can suppress downstream Aktsignaling and induce FoxO3A mediated cell death in SK cells but not innormal keratinocytes. Thus, essentially any small molecule inhibitor ofAkt signaling can be used for the pharmacological treatment of SKs.

Example 3 Inhibition of Akt Signaling Causes Cell Death in SK CellsThrough a FoxO3/p53 Mediated Mechanism

It is well-established that activation of Akt in cells is known toelicit pro-survival effects through activation/suppression of differenttargeted pathways. One of the common mechanisms for cell death upon Aktinhibition is induction of apoptosis, which was also observed upontreatment of SK cells with C8 as detected by cleaved PARP levels andpositive TUNEL staining (FIG. 8A, 8B).

Therefore it was investigated if any of the prominent pro-apoptotictargets of Akt were involved in this process. A decrease ofphosphorylated levels of FoxO1/3 as well as of phosphorylated MDM2 wasobserved, which correlated with increased p53 levels upon treatment withthe Akt inhibitor (FIG. 8C). Importantly when FoxO3 expression (a directAkt target³⁰) and/or p53 (an indirect Akt target,^(31, 32)) was depletedthrough siRNA in primary SK cells, the inventors were able to rescue thekilling effect of the Akt inhibitor in primary SK cells. These dataindicate that inhibition of Akt mediates a decrease of FoxO3phosphorylation and concomitant increase of p53 levels possibly throughrelease from MDM2^(31, 32), which eventually promotes apoptosis (FIG. 9)

Example 4 Exemplary Protocol for Culturing Seborrheic Keratosis Cells

Provided herein is the following exemplary method for culturing SK cellsfrom a biological sample. This method should not be construed aslimiting the invention to the following protocol.

Culturing SK Cells:

-   -   1. Collect the SK specimens in sterile DPBS, containing        Penicillin-Streptomycin (GIBCO, 15140-122). Keep at 40.    -   2. Prepare Pre-Dispase Solution:        -   250 ml Hanks Balanced Salt Solution—HBSS ((Gibco 14170-088)        -   2.5 ml filtered 1.0 M HEPES (GIBCO, 15630)        -   2.5 ml filtered 7.5% sodium bicarbonate    -   3. Make Dispase Solution:        -   10 mg/ml dispase in pre-dispase solution and filter with 0.2            micron filter    -   4. Place the SK pieces flat into suitable container (usually 12        well plate) facing up. Add approximately 0.5 ml dispase solution        around the SKs until they are floating.    -   5. Place in 4 C for at least 18 h (up to 24) THIS IS CRITICAL

Next day:

-   -   6. Add 5 ml Trypsin-EDTA to 50 ml falcon tube and warm it to        27 C. Take the SK pieces form the dispase and put them into the        Trypsin-EDTA. Place the tube into the incubator to keep at 37 C        for 5-7 min. Several times, during that period, carefully vortex        the tube.    -   7. Add 10 ml DMEM+10% FBS into the tube, mix and use cell        strainer to remove the bigger particles left.    -   8. Centrifuge at 1000 RPM for 10 min.    -   9. Resuspend the cells in HKC medium and plate on        collagen-coated plates.    -   10. Change the medium every other day.

These studies indicate the dependency of benign epidermal tumors onactivation of Akt signaling for survival. The experiments describedherein are designed to reveal how previously described genomicalterations in SKs may contribute to the activation of this pathway aswell as to identify its downstream targets responsible for thetransduction of pro-survival effects.

Recent reports indicate that epidermal SKs commonly harbor somaticmutations in key oncogenes such FGFR3, PIK3CA, HRAS, EGFR andAKT1^(5, 7, 33). These alterations represent the direct regulation oftwo essential molecular pathways in keratinocytes: the ERK/MAPK and PI3Ksignaling cascades. Similarly, the inventors' previous studies into thepathogenesis of SKs also identified aberrations upstream of the samesignaling pathways including overexpression of various receptor tyrosinekinases, growth hormones and transcription factors⁸. Taken togetherthese data indicate that the ERK/MAPK and PI3K pathways are responsiblefor the benign tumor phenotype of these lesions, characterized withenhanced growth, and suppressed apoptotic response/increased survival⁷.The data described herein indicate that despite the variety of genomicalterations in SKs, the growth dependency of SK cells converges toactivated Akt signaling. This hypothesis is further supported byevidence for increased phosphorylation of Akt in SK patient samplesharboring different mutations, when compared to their neighboringhealthy epidermis (FIG. 10). Moreover, while p53 mutations were notdetected in SKs⁷, the suppressed apoptotic response of these lesionsmight partially be due to the decreased levels of the indirect Akteffector, p53 in the same patient samples (FIG. 10).

Further data described herein indicate that inhibition of GSK3βphosphorylation in primary SK cells is a strong predictor foreffectiveness of Akt inhibitors (FIG. 11).

REFERENCES CITED

Each of the following references is incorporated herein by reference inits entirety.

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The invention claimed is:
 1. A method for screening a candidate agentfor inducing apoptosis, the method comprising: (a) contacting aseborrheic keratosis cell or population of seborrheic keratosis cellswith a candidate agent, and (b) measuring apoptosis in the cell orpopulation of cells, wherein an increase in apoptosis in the cell orpopulation of cells indicates that the candidate agent inducesapoptosis.
 2. The method of claim 1, wherein the candidate agentcomprises an Akt signaling pathway inhibitor.
 3. The method of claim 1,wherein the seborrheic keratosis cell or population of seborrheickeratosis cells are cultured using a method comprising the steps of: (a)contacting a biological sample comprising seborrheic keratosis cellsobtained from a subject with a solution comprising a dispase enzyme at atemperature and for a time sufficient to initiate dissociation of theseborrheic keratosis cells from the biological sample, and (b) culturingthe dissociated seborrheic keratosis cells.
 4. The method of claim 1,wherein apoptosis is measured using sulforhodamine B (SRB) assay, MTTtetrazolium dye, TUNEL staining, Annexin V staining, propidium iodidestaining, DNA laddering, PARP cleavage, caspase activation, and/orassessment of cellular and nuclear morphology.
 5. The method of claim 1,wherein the candidate agent is a small molecule, a peptide inhibitor, oran RNAi molecule.
 6. An assay comprising: (a) contacting a population ofdissociated seborrheic keratosis cells with a candidate agent, (b)contacting the cells of step (a) with a detectable antibody specific foran apoptotic protein, (c) measuring the intensity of the signal from thebound, detectable antibody, (d) comparing the measured intensity of thesignal with a reference value and if the measured intensity is increasedrelative to the reference value, (e) identifying the candidate agent asan inducer of apoptosis in the cell.
 7. The assay of claim 6, whereinthe candidate agent comprises an Akt signaling pathway inhibitor.
 8. Theassay of claim 6, wherein the population of seborrheic keratosis cellsis cultured using a method comprising the steps of: (a) contacting abiological sample comprising seborrheic keratosis cells obtained from asubject with a solution comprising a dispase enzyme at a temperature andfor a time sufficient to initiate dissociation of the seborrheickeratosis cells from the biological sample, and (b) culturing thedissociated seborrheic keratosis cells.
 9. The assay of claim 6, whereinthe apoptotic protein is a caspase protein, a PARP protein, or acleavage product thereof.